THE JOURNAL OF BIOLOGICAL CHEMISTRY
7264 –7274, 2004
© 2004 by The American Society for Biochemistry and Molecular Biology, Inc.
Intermedin Is a Calcitonin/Calcitonin Gene-related Peptide Family
Peptide Acting through the Calcitonin Receptor-like
Receptor/Receptor Activity-modifying Protein Receptor Complexes*
Received for publication, May 21, 2003, and in revised form, October 15, 2003
Published, JBC Papers in Press, November 13, 2003, DOI 10.1074/jbc.M305332200
Jaesook Roh, Chia Lin Chang‡, Alka Bhalla, Cynthia Klein, and Sheau Yu Teddy Hsu§
From the Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School
of Medicine, Stanford, California 94305-5317
Calcitonin, calcitonin gene-related peptide (CGRP),
adrenomedullin (ADM), and amylin belong to a unique
group of peptide hormones important for homeostasis in
diverse tissues. Calcitonin is essential for calcium bal-
ance, whereas CGRP and ADM are important for neuro-
transmission and cardiovascular and respiratory regu-
lation. Based on phylogenetic analysis, we identified
intermedin as a novel member of the calcitonin/CGRP
peptide family. Analysis of intermedin expression indi-
cated that intermedin is expressed primarily in the pi-
tuitary and gastrointestinal tract. Intermedin increased
cAMP production in SK-N-MC and L6 cells expressing
endogenous CGRP receptors and competed with labeled
CGRP for binding to its receptors in these cells. In ad-
dition, treatment of 293T cells expressing recombinant
calcitonin receptor-like receptor (CRLR) and one of the
three receptor activity-modifying proteins (RAMPs)
showed that a CRLR/RAMP receptor complex is re-
quired for intermedin signaling. In contrast to CGRP
and ADM, which exhibited a preferential stimulation of
CRLR when co-expressed with RAMP1 and RAMP2 or
RAMP3, respectively, intermedin represents a nonselec-
tive agonist for the RAMP coreceptors. In vivo studies
demonstrated that intermedin treatment led to blood
pressure reduction in both normal and spontaneously
hypertensive rats via interactions with the CRLR/RAMP
receptor complexes. Furthermore, in vivo treatment in
mice with intermedin led to suppression of gastric emp-
tying activity and food intake. Thus, identification of
intermedin as a novel member of the calcitonin/CGRP
peptide family capable of signaling through CRLR/
RAMP receptor complexes provides an additional
player in the regulation of peripheral tissues by CRLR
and will allow development of new therapeutic agents
for pathologies associated with diverse vascular and
gastrointestinal disorders.
Originally isolated as a polypeptide hormone essential for
calcium balance (1, 2), calcitonin belongs to a group of peptide
* The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted
to the GenBankTM/EBI Data Bank with accession number(s) AF529213.
‡ Supported by a Dean’s fellowship from the Department of Obstet-
rics and Gynecology, Chang Gung Memorial Hospital (Tao-Yuan,
Taiwan).
§ To whom correspondence should be addressed: Dept. of Obstetrics
and Gynecology, Stanford University School of Medicine, A344E, 300
Pasteur Dr., Stanford, CA 94305-5317. Tel.: 650-723-7057; Fax: 650-
725-7102; E-mail: teddyhsu@stanford.edu.
7264
Vol. 279, No. 8, Issue of February 20, pp.
Printed in U.S.A.
hormones including ␣-CGRP,1 ␤-CGRP, adrenomedullin
(ADM), and amylin (3). Among these tissue-specific peptides,
ADM and CGRP are important endocrine and neurocrine inte-
grators of homeostasis in the vascular and respiratory systems,
whereas amylin is essential for optimal glucose metabolism.
The biological actions of these peptides are mediated via bind-
ing to two closely related type II G protein-coupled receptors
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(GPCRs), the calcitonin receptor, and the calcitonin receptor-
like receptor (CRLR) (4, 5). Although the calcitonin receptor is
the main mediator for calcitonin action, it also binds amylin.
Recent cloning and functional studies have shown that CGRP,
ADM, and, to a lesser extent, amylin interact with different
combinations of CRLR and the three receptor activity-modify-
ing proteins (RAMPs) (5, 6). Studies using mutant mice defi-
cient in ␣-CGRP, ADM, or amylin have indicated that CRLR
could be important for cardiovascular morphogenesis, sensory
neurotransmission, inflammatory reactions, nociceptive behav-
ior, and glucose homeostasis (7–12). Thus, the physiological
functions of the peptides in this family are determined by
receptor-binding specificity and the tissue expression profiles
of individual ligands.
Because the expression of CGRP and its binding sites does
not overlap in the brain (13), we hypothesized the existence of
additional calcitonin/CGRP family peptides. Using a phyloge-
netic profiling approach to analyze the GenBankTM/EBI Data
Bank, we identified a novel calcitonin/CGRP family peptide,
intermedin, from the genomes of human and other vertebrates.
Sequence analysis of the prepropolypeptides of different family
genes indicated that the sequence homology between interme-
din and the paralogous peptides is restricted to the mature
peptide and that the intermedin gene evolved during early
vertebrate evolution. Pharmacological analyses showed that
intermedin signals through CRLR/RAMP receptor complexes
and activates the cAMP-dependent pathway in transfected
cells. We show that intermedin signals through the CRLR
signaling system to regulate vascular and gastrointestinal
functions in vivo.
EXPERIMENTAL PROCEDURES
Cloning, Phylogenetic Analysis, and Expression Profiles of Human
Intermedin—Human intermedin was initially identified from an ex-
pressed sequence tag and a genomic sequence (AK024788 and
AL096767 in GenBank™), and its identity was verified by PCR ampli-
fication using a human Marathon-Ready pituitary cDNA library (Clon-
tech). For analysis of intermedin mRNAs in the human digestive sys-
1
The abbreviations used are: CGRP, calcitonin gene-related peptide;
ADM, adrenomedullin; GPCR, G protein-coupled receptor; CRLR, cal-
citonin receptor-like receptor; RAMP, receptor activity-modifying pro-
tein; SHRs, spontaneously hypertensive rats; IMD, intermedin; IMDL,
intermedin-long; IMDS, intermedin-short; SRP, stresscopin-related
peptide; MSH, melanin-stimulating hormone.
This paper is available on line at http://www.jbc.org
 Intermedin Is a Novel Calcitonin/CGRP Family Peptide
tem, normalized first strand cDNA preparations were obtained from
Clontech. The putative intermedin peptides from fish were deduced
based on a zebrafish expressed sequence tag sequence (AW421384) and
puffer fish genomic sequences (Fugu rubripes; Scaffold_1011). The rat
and mouse intermedin sequences were deduced based on expressed
sequence tags BQ192607 and BG918210, respectively. Putative puffer
fish ␣-CGRP, ␤-CGRP, ADM, and amylin sequences were deduced
based on puffer fish sequences Scaffold_9445, Scaffold_6549,
JGI_28042, and JGI_8403, respectively.2 The BLOCK MAKER pro-
gram3 was used to align the mature peptides from different species.
Phylogenetic analysis was carried out using a routine in ClustalW.4 The
consensus secondary structure of calcitonin/CGRP family peptides was
predicted using the Network Protein Sequence Analysis server.5 For
Northern blot analysis of intermedin expression, pituitary RNAs were
extracted from pituitary glands obtained from male Sprague-Dawley
rats. Following extraction using TRIzol solution, total RNA was re-
solved on formaldehyde-agarose gels and hybridized with a 32P-labeled
rat intermedin cDNA probe. The x-ray film was exposed at ⫺80 °C for
1 week with intensifying screens.
Peptide Synthesis—Intermedin-related peptides were synthesized
based on the solid-phase Fmoc (N-(9-fluorenyl)methoxycarbonyl) proto-
col and analyzed by reverse-phase high pressure liquid chromatography
with a Vydac C18 analytical column and by mass spectrometry using a
matrix-assisted laser desorption ionization time-of-flight Voyager-DE
RP workstation. Synthetic ADM, ␤-CGRP, and related peptides were
obtained from Sigma), AnaSpec, Inc. (San Jose, CA), and Bachem (Tor-
rance, CA). Radiolabeled 125I-CGRP (2000 Ci/mmol) was from Amer-
sham Biosciences. Stocks of different hormones were prepared in dis-
tilled water and diluted in culture medium.
Immunoanalysis—Rabbit anti-intermedin antibodies were generated
using synthetic peptides corresponding to residues 28 – 47 (MGPAGRQD-
SAPVDPSSPHSY) of human intermedin (Strategic Biosolutions,
Ramona, CA). This peptide antigen was selected based on the high se-
quence identity (85%) found in this region of human and rodent interme-
dins and the negligible similarity to other family peptides. The intermedin
peptide was conjugated to keyhole limpet hemocyanin using 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide hydrochloride before immunization.
Antibodies were purified using antigen-conjugated affinity columns. In
immunoblot analysis, the anti-human intermedin antibody cross-reacted
with synthetic intermedin counterparts from different vertebrates, but
not with paralogous peptides, including calcitonin, CGRP, ADM, and
amylin. For immunohistochemical analysis, tissues were obtained from
adult rats, mice, and bullfrogs and analyzed as described (14). To demon-
strate that the intermedin transcript encodes the predicted intermedin
mature peptide, full-length human intermedin cDNA was subcloned into
the pcDNA3.1 expression vector. For Western blot analysis of intermedin
in culture medium, 293T cells were transfected with the intermedin
expression vector using the calcium phosphate precipitation method. For-
ty-eight hours after transfection, the serum-free culture medium was
harvested and concentrated using a Centricon-3 column. After concentra-
tion, the supernatant was boiled for 5 min in denaturing buffer with 100
mM dithiothreitol before SDS-PAGE and Western blot analysis using
anti-intermedin antibodies.
Stimulation of cAMP Production in SK-N-MC and L6 Cells by Inter-
medin and Related Peptides—Human neuroblastoma SK-N-MC cells
and rat L6 skeletal myoblast cells expressing endogenous CRLR were
obtained from American Type Culture Collection. To estimate adenylyl
cyclase activation, SK-N-MC and L6 cells (2 ⫻ 105 viable cells/well)
were plated in 24-well culture dishes in Dulbecco’s modified Eagle’s
medium/nutrient mixture F-12 1 day before treatment. Following a 2-h
incubation in serum-free Dulbecco’s modified Eagle’s medium/nutrient
mixture F-12, cells were treated with testing reagents for 30 min in
medium containing 0.1% bovine serum albumin and 2.5 mM 3-isobutyl-
1-methylxanthine (Sigma) to prevent hydrolysis of cAMP by phosphodi-
esterases. Following treatment, cells were lysed, and cAMP content was
determined by a specific radioimmunoassay (15).
Activation of CRLR/RAMP Receptor Complexes by Intermedin in
Transfected 293T Cells—To study the interaction between intermedin
and the CRLR/RAMP receptor complexes, we cloned human CRLR,
RAMP1, RAMP2, and RAMP3 (accession numbers NP_005786,
NP_005846, NP_005845, and O60896, respectively) cDNAs by PCR
from human Marathon-Ready cDNA libraries using two sets of primers
2
Available at www.jgi.doe.gov/fugu/index.html.
3
Available at blocks.fhcrc.org.
4
Available at blocks.fhcrc.org/blocks.
5
Available at pbil.ibcp.fr/.
7265
flanking the full-length coding sequences of each gene. Each cDNA was
verified by DNA sequencing and subcloned into the pcDNA3.1 expres-
sion vector. To allow the detection of cell-surface expression of these
proteins, CRLR and RAMPs were tagged at the N terminus of the
mature protein with a FLAG epitope. Because it has been shown that
epitope tagging affects the RAMP1 protein signaling (16), we used the
wild-type RAMP1 construct for analysis of intermedin signaling.
HEK293T cells were maintained in 35-mm culture dishes in Dulbecco’s
modified Eagle’s medium/Ham’s F-12 medium (Invitrogen) supple-
mented with 10% fetal bovine serum, 100 ␮g/ml penicillin, 100 ␮g/ml
streptomycin, and 2 mM L-glutamine. The cells were cotransfected with
10 ␮g of CRLR and/or 10 ␮g of RAMP expression plasmid using the
calcium phosphate precipitation method. Forty-eight hours after trans-
fection, cells were washed twice with Dulbecco’s phosphate-buffered
saline, harvested from culture dishes, and centrifuged at 400 ⫻ g for 5
min. To determine the level of expression of CRLR and RAMP on the
cell surface, the resuspended cells (2 ⫻ 106/tube) were incubated with
anti-FLAG antibody M1 (50 mg/ml; Sigma) in Tris-buffered saline (pH
7.4) containing 5 mg/ml bovine serum albumin and 2 mM CaCl2 (assay
buffer) for 4 h at room temperature in siliconized tubes. Cells were then
washed twice with 1 ml of assay buffer after centrifugation at 14,000 ⫻
g for 15 s. The horseradish peroxidase-conjugated secondary antibody
(sheep anti-mouse IgG) was added to the resuspended cell pellets and
incubated for 1 h at room temperature. Cells were washed twice with 1
ml of assay buffer by repeated centrifugation before determination of
horseradish peroxidase activity in cell pellets using ECL reagents (Am-
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ersham Biosciences) and a Lumimark microplate reader (Bio-Rad).
Background binding was determined by adding excess amounts of the
synthetic FLAG peptide (Sigma) at a concentration of 100 ␮g/ml. For
the assay of adenylyl cyclase activation in transfected cells, cells (2 ⫻
105/ml) were placed in 24-well tissue culture plates (Corning Inc.,
Corning, NY) and preincubated at 37 °C for 30 min in the presence of
2.5 mM 3-isobutyl-1-methylxanthine before hormonal treatment for 4 h.
Receptor Binding Assay—Ligand binding assays were done in sili-
conized microcentrifuge tubes at 37 °C for 2 h. Intact SK-N-MC and L6
cells were resuspended in binding buffer (20 mM Tris-HCl (pH 7.4), 2
mM MgCl2, and 0.1% bovine serum albumin) with 0.06 ␮g of 125I-CGRP
and various concentrations of nonradioactive peptides. After a 2-h
incubation at 37 °C, the cell-associated ligand was estimated (15).
Radioactivity was determined using a ␥-counter (EG&G Wallace,
Gaithersburg, MD).
Effects of Intermedin on Blood Pressure and Heart Rate in Normal
and Hypertensive Rats—Blood pressure measurements were made in
conscious male Sprague-Dawley rats and spontaneously hypertensive
rats (SHRs; 7–9 weeks of age) pre-adapted to the measurement proce-
dure. Indirect systolic pressure was determined by a programmable
non-invasive blood pressure system using the tail-cuff method (Colum-
bus Instruments, Columbus, OH). Following attachment of the pres-
sure transducer, rats were left undisturbed for 10 min before base-line
measurements that spanned a 15-min interval. Following base-line
measurements, rats were injected intraperitoneally with varying doses
of hormones. Blood pressure and heart rate were monitored for 40 min
at 20-s intervals. Changes in blood pressure were calculated as the
average of 30 measurements performed within each 10-min interval.
Effects of Intermedin on Gastric Emptying Activity—Eight-week-old
male C57BL/6 mice deprived of food for 20 h were given food pellets for
90 min before intraperitoneal injection with different hormones or
saline. After treatment, mice were deprived of food again and killed 90
min later. The stomach was excised at the pylorus and cardia before
weighing. Gastric emptying was calculated by comparing the stomach
weight of treated mice with the stomach weight of control mice killed at
the time of hormone injection.
Analysis of Ingestive Behavior—Eight-week-old male C57BL/6 mice
were housed individually in a regulated environment. Before intraperi-
toneal injection with testing reagents, mice were deprived of food for
20 h with free access to water. Food intake was measured by placing
pre-weighed pellets in the cage and weighing uneaten pellets at 1, 2,
and 4 h after treatment.
Statistical Analysis—Differences between treatment groups were an-
alyzed by analysis of variance and Student’s t test.
RESULTS
Intermedin as a Calcitonin/CGRP Family Peptide—We
searched the GenBankTM/EBI Data Bank for sequence motifs
with unique primary and secondary structures shared by all
calcitonin/CGRP family peptides using a phylogenetic profiling
approach that has been used to identify novel corticotropin-
7266 Intermedin Is a Novel Calcitonin/CGRP Family Peptide
releasing hormone family peptides (15). Candidate sequences
were screened for the presence of proteolytic cleavage sites
flanking the putative mature region of the precursor proteins.
Based on these criteria, we identified intermedin genes from
mammals and teleosts, including zebrafish and a Japanese
puffer fish (Takifugu rubripes). Human intermedin encodes a
preproprotein of 148 amino acids with a signal peptide for
secretion at the N terminus (Fig. 1A). Although the overall
amino acid sequence of intermedin shows no similarity to
known proteins, a stretch of 47 residues at the C terminus is
flanked by dibasic proteolytic cleavage sites at the N terminus
and an ␣-amidation donor residue at the C terminus. The
putative mature region of intermedin shares ⬃28% sequence
identity with ADM and ⬍20% with CGRP (Fig. 1B). Impor-
tantly, the predicted mature region adopts an N-terminal dis-
ulfide-bonded loop leading into an ␣-helix, followed by a disor-
dered structure that is shared by all calcitonin/CGRP family
peptides (Fig. 1B). Furthermore, sequence alignment of inter-
medin precursors from mammals and teleosts indicated that
sequence conservation in orthologous intermedins is restricted
to the mature region. The mature intermedins of human and
fish share ⬎60% similarity, whereas human and rodent inter-
medins are 87% identical (Fig. 1B). In addition, the mouse and
rat intermedin peptides appear to differ by only one amino acid.
Furthermore, analysis of orthologous intermedins indicated
that the positions of N-terminal dibasic cleavage sites vary by
a few amino acids among different species, whereas an arginine
residue seven amino acids downstream of the dibasic cleavage
motif of human intermedin is conserved in all species, suggest-
ing that the mature intermedin from human and other species
could be a 40-amino acid peptide. On the basis of these se-
quence analyses, we predicted that a 47-amino acid mature
peptide (intermedin-long (IMDL)) and a shorter 40-amino acid
intermedin (intermedin-short (IMDS)) could be generated by
proteolytic cleavage at the N-terminal proximate basic residues
followed by an amidated C terminus. Because the putative
preproregion of intermedin from diverse vertebrates is not con-
served, intermedin is unlikely to encode additional active pep-
tides such as the pro-ADM N-terminal 20-amino acid peptide
found in the ADM precursor (17).
Phylogenetic analysis of 12 CGRP family peptides from fish
and mammals suggested an ancient evolution for three sub-
groups of these peptide hormones, with mammalian and teleost
intermedins clustered in a separate branch with ADM and
CGRP (Fig. 1C). Thus, intermedin and other family peptides
evolved before the emergence of modern teleosts and tetrapods.
Genomic analysis showed that intermedin is located on the
distal arm of human chromosome 22q13 and syntenic mouse
chromosome 15. In both human and mouse genomes, interme-
din neighbors an aldehyde reductase-like gene. In contrast, all
other calcitonin/CGRP family genes cluster on human chromo-
somes 11 and 12.
Intermedin Activates the cAMP-dependent Pathway in SK-
N-MC and L6 Cells via the CGRP Receptor—Pairwise sequence
comparison and phylogenetic tree building based on all GPCR
sequences indicated that intermedin is closest to ADM and
CGRP, whereas no orphan GPCR shares a close relatedness
with CRLR, the receptor for ADM and CGRP. Thus, CRLR is a
candidate receptor for intermedin. To test this hypothesis, we
treated human neuroblastoma SK-N-MC cells and rat L6 skel-
etal myoblast cells, known to express different levels of CRLR
and RAMPs (18), with synthetic intermedin peptides and then
monitored cAMP production. As shown in Fig. 2 (A and B),
treatment with the amidated IMDL (amino acids 1– 47) or
IMDS (amino acids 8 – 47) peptide resulted in dose-dependent
increases in cAMP production in both cell lines. The observed
activation is specific, as treatment with a nonamidated form of
intermedin, a truncated amidated intermedin fragment (inter-
medin (IMD)-(17– 47)), or a 31-amino acid peptide from the
preproregion of human intermedin (prointermedin-(55– 85))
had no effect in either cell line (data not shown), suggesting
that ␣-amidation and residues 8 –16 of intermedin are impor-
tant for intermedin bioactivity. Consistent with earlier reports
(5, 18, 19), both ADM and ␤-CGRP also stimulated cAMP
production in these cell lines (Fig. 2, A and B). Of importance,
the stimulatory effect of intermedin was suppressed by co-
treatment with a CGRP receptor antagonist (CGRP-(8 –37)) in
L6 cells (Fig. 2C), demonstrating that intermedin activates the
cAMP-dependent pathway via the CGRP receptor (20). To fur-
ther characterize the specific action of intermedin on cAMP
production, L6 cells were co-treated with a putative intermedin
C-terminal receptor-binding domain (IMD-(17– 47)) or an anti-
intermedin polyclonal antibody. As shown in Fig. 2D, IMD-(17–
47) was found to be a functional antagonist of intermedin
action, consistent with the observed antagonistic effect of N-
terminally truncated CGRP-(8 –37) (Fig. 2C) (20). In addition,
co-treatment with the anti-intermedin antibody blocked the
stimulatory effect of intermedin, whereas co-treatment with an
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antibody raised against the unrelated stresscopin-related pep-
tide (SRP)/urocortin II had no effect (Fig. 2D).
To establish a direct interaction between intermedin and
CRLR, we used iodinated CGRP as the radioligand for receptor
binding assays. As shown in Fig. 2 (E and F), IMDL and IMDS
displaced 125I-CGRP binding to the SK-N-MC and L6 cells
dose-dependently.
Intermedin Is a Nonselective Agonist for CRLR/RAMP Recep-
tor Complexes—CGRP and adrenomedullin mediate their ac-
tion through the CRLR/RAMP complexes, consisting of CRLR
and one of the three RAMP polypeptides. To investigate the
role of CRLR/RAMP receptor complexes in intermedin signal-
ing, we treated 293T cells expressing different combinations of
recombinant CRLR and/or RAMPs with intermedin and related
peptides. As shown in Fig. 3A, treatment of intermedin, CGRP,
or ADM had no effect on cAMP production in 293T cells ex-
pressing CRLR alone, whereas calcitonin increased cAMP pro-
duction dose-dependently via the endogenous calcitonin recep-
tor. In contrast, intermedin dose-dependently stimulated
cAMP production in cells expressing different CRLR/RAMP
receptor complexes (Fig. 3, B–D). Consistent with earlier stud-
ies, CGRP and ADM exhibited a preferential stimulation of
CRLR when co-expressed with RAMP1 and RAMP2 or RAMP3,
respectively. Compared with CGRP, intermedin exhibited a
greater potency in the stimulation of cAMP production in cells
expressing CRLR/RAMP3, but had lower activity with CRLR/
RAMP1. In contrast, intermedin had a lower potency in the
activation of both CRLR/RAMP2 and CRLR/RAMP3 compared
with adrenomedullin. Thus, the overall rank of potency for the
stimulation of CRLR/RAMP1, CRLR/RAMP2, and CRLR/
RAMP3 is CGRP ⬎ IMD ⫽ ADM, ADM ⬎ IMD ⫽ CGRP, and
ADM ⬎ IMD ⬎ CGRP, respectively. Furthermore, consistent
with earlier reports, the expression of CRLR/RAMP receptor
complexes on the cell surface of transfected cells was found to
be increased synergistically by co-expressing CRLR and
RAMPs (Fig. 3E).
Intermedin Expression in the Pituitary and Stomach—Initial
reverse transcription-PCR analysis showed that the interme-
din transcript is expressed in the pituitary and stomach.
Northern blot analysis of rat pituitary RNA showed that two
specific intermedin transcripts of ⬃5 and 2.5 kb were present
in the pituitary (Fig. 4A). To further characterize the expres-
sion profile of intermedin, four independent antibodies were
developed using a C-terminal 20-amino acid intermedin pep-
 Intermedin Is a Novel Calcitonin/CGRP Family Peptide 7267

FIG. 1. Cloning of intermedin and

elucidation of its identity. A, the hu-
man intermedin gene encodes a 148-a-
mino acid open reading frame with a 24-
amino acid signal peptide for secretion at
the N terminus. Amino acid positions are
on the right, and the stop codon is marked
with an asterisk. The putative mature
peptide is underlined, and the N-terminal
signal peptide for secretion is lightly
shaded. The ATG start site and the puta-
tive C-terminal amidation donor residue
are in boldface. The putative basic cleav-

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age sites are darkly shaded. B, compari-
son of CGRP-related peptides (␣-CGRP,
␤-CGRP, amylin, ADM, and IMD) from
mammals and fish. Sequence alignment
of the precursors of these peptides from
human indicated that the sequence ho-
mology between intermedin and paralo-
gous peptides is restricted to the mature
peptide, and no similarity was found in
the putative preproregion. The two inter-
medins from puffer fish (T. rubripes) are
indicated as IMD1 and IMD2, respec-
tively. The putative secondary structures
of mature CGRP peptides are indicated
above the alignment, and the putative
secondary structures of mature interme-
dins are shown below the alignment. In
addition, the predicted IMDL and IMDS
peptides are indicated by dotted lines un-
derneath the alignment. Curved lines,
random coil; round cylinder, extended
strand; wavy banners, helix. The two cys-
teines and one neighboring threonine res-
idue shared by all aligned peptides are
boxed. Residues shared by ADM and in-
termedin from different vertebrates are
indicated by asterisks. Residues shared by
highly conserved orthologous sequences
of each gene are lightly shaded. The N-
terminal arginine residue found in all in-
termedins from different species is shown
in boldface. h, human; m, mouse; p, puffer
fish; r, rat; z, zebrafish. C, the phyloge-
netic relationship among 12 representa-
tive CGRP-related peptides.

tide (IMD-(28 – 47)). As shown in Fig. 4B, the anti-intermedin antibody, immunohistochemical analysis of ⬎20 different
antibody (C2411-2) is specific for intermedin and showed no mouse tissues confirmed intermedin expression in the pituitary
cross-reaction with related peptides, including calcitonin, and stomach. As shown in Fig. 4 (C (magnification ⫻100) and
CGRP, ADM, and amylin. Using the specific anti-intermedin D (magnification ⫻200)), intermedin was expressed mainly in
7268 Intermedin Is a Novel Calcitonin/CGRP Family Peptide

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FIG. 2. Intermedin shares receptors with CGRP and ADM. A and B, synthetic intermedin peptides (IMDL and IMDS) stimulate cAMP
production in human neuroblastoma SK-N-MC cells and rat L6 skeletal myoblast cells, respectively. No stimulation by a nonamidated form of
intermedin, a truncated amidated intermedin fragment (IMD-(17– 47), or a 31-amino acid peptide from the preproregion of intermedin
(prointermedin-(55– 85)) was observed (data not shown). Data are means ⫾ S.E. (n ⫽ 4). C, blockage of the stimulatory effect of intermedin on
cAMP production by a CGRP receptor antagonist (CGRP-(8 –37)) in L6 cells. D, blockage of the stimulatory effect of intermedin by IMD-(17– 47)
(1 ␮M) and the anti-intermedin antibody (anti-IMD Ab) in L6 cells. Data are means ⫾ S.E. (n ⫽ 4). No effect was observed upon co-treatment with
an anti-SRP/urocortin II antibody (anti-SRP Ab). *, significantly different from controls (p ⬍ 0.05). E and F, competitive displacement by unlabeled
intermedin and related peptides of 125I-CGRP bound to SK-N-MC and L6 cells, respectively. Data are means ⫾ S.E. (n ⫽ 3).

the intermediate lobe of the pituitary, with sporadic signals in
the anterior lobe. In contrast, negative controls using preim-
mune serum or anti-intermedin antibodies presaturated with
the intermedin antigen showed no specific signals (Fig. 4, E
and F). Likewise, immunohistochemical analysis of pituitary
sections from rats and bullfrogs showed that intermedin ex-
pression was restricted to the intermediate and anterior lobes
of the pituitary (Fig. 4, G and H). Because melanin-stimulating
hormone (MSH) had a similar expression pattern in the pitui-
tary (Fig. 4I, anti-MSH staining), we tested whether the anti-
intermedin antibody cross-reacts with the MSH peptide. As
shown in Fig. 4J, the specific staining of intermedin in the
pituitary was not abolished by preincubation with an MSH
peptide. To further demonstrate that the intermedin mRNA
encodes the predicted mature intermedin peptide, a human
intermedin cDNA was subcloned in the eukaryotic expression
vector pcDNA3.1, and the expression of intermedin peptide
from this construct was investigated using transfected 293T
cells. Western blot analysis of concentrated culture media
showed that cells transfected with the intermedin expression
vector secreted an ⬃5-kDa mature intermedin peptide into the
culture medium, whereas the culture medium from cells trans-
 Intermedin Is a Novel Calcitonin/CGRP Family Peptide 7269

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FIG. 3. Intermedin activates recombinant CRLR/RAMP receptor complexes in transiently transfected 293T cells. Treatment of 293T
cells transiently transfected with an empty expression vector or a CRLR expression vector with intermedin, CGRP, or ADM had no effect on whole
cell cAMP production (A). In contrast, calcitonin increased cAMP production dose-dependently through the endogenous calcitonin receptor. Unlike
cells expressing CRLR solely, treatment with intermedin increased cAMP production in cells expressing CRLR with RAMP1 (B), RAMP2 (C), or
RAMP3 (D). Likewise, treatment with CGRP or ADM stimulated cAMP production in cells expressing CRLR/RAMP receptor complexes with
different potency (B–D; n ⫽ 3). Shown are the results of indirect binding analysis of cell-surface expression of CRLR and RAMPs using horseradish
peroxidase-conjugated sheep anti-mouse antibodies and anti-FLAG epitope antibodies (E). Expression of FLAG epitope-tagged CRLR and RAMPs
on the cell surface of transfected cells was increased by co-transfection with CRLR and RAMP expression vectors compared with transfection with
a single expression vector encoding CRLR or RAMP.

fected with the empty vector displayed no signal (Fig. 4K).
To characterize the expression of intermedin in the gastro-
intestinal tract, a panel of human cDNAs from the gastrointes-
tinal tract was analyzed by PCR. As shown in Fig. 5A (first
panel, 1 ng of cDNA template/tube), the expression of the
intermedin transcript could be detected in the esophagus,
stomach, jejunum, ileum, ileocecum, ascending colon, trans-
verse colon, descending colon, and rectum. PCR analysis using
a lower amount of cDNA templates (10 pg/tube) showed that
the expression of the intermedin transcript was greater in the
stomach and jejunum (Fig. 5A, second panel). Furthermore,
immunohistochemical staining showed that intermedin was
found primarily in the muscularis mucosae layer of the stom-
ach (Fig. 5B) and that the signal was abolished by presatura-
tion with the intermedin antigen (Fig. 5C).
Systemic Hypotensive Action of Intermedin—Because the re-
lated ADM is one of the most potent vasodilators (5) and the
pituitary-derived intermedin could be released into the sys-
temic circulation to act on diverse peripheral tissues, we tested
the effect of intermedin on blood pressure regulation in normal
7270 Intermedin Is a Novel Calcitonin/CGRP Family Peptide

FIG. 4. Expression of intermedin in

the pituitary. A, Northern blot analysis
showing that two specific intermedin
transcripts were expressed in rat pitui-
tary cells. The positions of 28 S and 18 S
RNAs are indicated. B, Western blot anal-
ysis of synthetic peptides using an anti-
intermedin antibody generated against
the C-terminal 20 amino acids of human
intermedin (MGPAGRQDSAPVDPSSP-
HSY). The anti-intermedin antibody is
specific for intermedin and showed no

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cross-reaction with CGRP, calcitonin,
ADM, or amylin. Molecular mass markers
are shown on the left, and specific bands
are indicated by arrows. C–F, immunohis-
tochemical staining of mouse pituitary
sections using the anti-intermedin anti-
body (C, magnification ⫻100; D, magnifi-
cation ⫻200), preimmune rabbit serum
(E), or anti-intermedin antibody presatu-
rated with the intermedin ligand (F). Sec-
tions incubated with preimmune serum
(E) or antibody presaturated with the in-
termedin peptide antigen (F) showed
negligible signals. G and H, immunohis-
tochemical analysis of intermedin expres-
sion in pituitary sections from rat and
bullfrog, respectively. I and J, immuno-
histochemical staining of mouse pituitary
sections using an anti-MSH antibody or
the anti-intermedin antibody presatu-
rated with an MSH peptide, respectively.
Specific signals are indicated by arrows.
AL, anterior lobe; IL, intermediate lobe;
PL, posterior lobe. K, Western blot anal-
ysis of concentrated culture medium from
293T cells transfected with an intermedin
expression vector. The anti-intermedin
antibody detected an ⬃5-kDa mature in-
termedin peptide in the culture medium,
whereas the culture medium from cells
transfected with the empty vector dis-
played no signal. Specific intermedin sig-
nals are indicated by the arrow. Positive
signals from the synthetic intermedin
peptide are shown in the left lanes.

rats and SHRs using a noninvasive monitoring approach. As Sprague-Dawley rats, similar to that induced by ADM. In ad-
shown in Fig. 6A, intraperitoneal administration of IMDL or dition, treatment of IMDL or IMDS also increased heart rate,
IMDS dose-dependently suppressed blood pressure in normal as found for ADM (Fig. 6B). In contrast, administration of the
 Intermedin Is a Novel Calcitonin/CGRP Family Peptide 7271
FIG. 5. Expression of intermedin in
digestive tissues. For analysis of inter-
medin mRNAs in the human digestive
system, normalized first strand cDNA
preparations from the human esophagus,
stomach, duodenum, jejunum, ileum, ileo-
cecum, cecum, ascending (Asc.) colon,
transverse (Trans.) colon, descending
(Des.) colon, and rectum (first panel, 1 ng of
template/reaction; second panel, 10 pg of
template/reaction) were obtained from
Clontech (A). Specific bands (303 bp) were
PCR-amplified using intermedin gene-
specific primer pairs under high stringency
conditions. The primer sequences for inter-
medin PCR analysis are 5⬘-AGGGAGGG-
GAACTCAGCAGTTCAGGAG-3⬘ (forward)
and 5⬘-GTTCTTGTTCTTGCTGTCACTT-
GGGCCT-3⬘ (reverse). Expression of glyc-
eraldehyde-3-phosphate dehydrogenase
(GAPDH) transcripts in different cDNA
templates was also analyzed to assess the
quality of the cDNA templates (third panel,
1 ng of template/reaction; fourth panel, 10
pg of template/reaction). Immunohisto-
chemical staining of mouse stomach sec-
tions showed that intermedin was found

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primarily in the muscularis mucosae layer
of the stomach (B), and the signal was abol-
ished by presaturation with the intermedin
antigen (C). Specific signals are indicated
by arrows. MU, mucosal layer; MS, muscu-
laris layer; SL, serosal layer.

truncated IMD-(17– 47) fragment (Fig. 6C) or the prointermedin- DISCUSSION

(55– 85) peptide (data not shown) had no effect on blood pres-
sure regulation. Because intermedin signals through CRLR/
RAMP receptor complexes, the ability of a CGRP receptor
antagonist (CGRP-(8 –37)) to block the actions of intermedin
was also studied. As shown in Fig. 6C, treatment with a 20-fold
excess of CGRP-(8 –37) significantly decreased the hypotensive
effects of IMDL. Likewise, co-treatment with the putative in-
termedin receptor-binding domain fragment IMD-(17– 47)
blocked the hypotensive effects of IMDL. In addition, we stud-
ied the hypotensive effect of intermedin in SHRs. IMDL treat-
ment reduced blood pressure in SHRs (similar to the effect in
normal rats), and the hypotensive effects of IMDL were abol-
ished by co-treatment with CGRP-(8 –37) (Fig. 6D). In contrast,
co-treatment with the low affinity ADM-(22–52) fragment had
a minimal effect (21). Thus, intermedin is a specific ligand for
the vascular CRLR/RAMP signaling system and could be im-
portant in the mediation of vascular responses for homeostasis.
Intermedin Suppresses Food Intake and Gastric Emptying—
Earlier studies have shown that both CGRP and ADM have
potent anorexic effects (22) and could mediate actions through
central or peripheral CRLR/RAMP systems. To examine
whether intermedin has a role in anorexia regulation, we stud-
ied the ability of intermedin to regulate feeding behavior based
on cumulative food intake in fasted mice. Intraperitoneal in-
jection with IMDL, IMDS, ADM, or a type II corticotropin-
releasing hormone receptor-selective agonist (SRP/urocortin II)
decreased food intake in fasted mice (Fig. 7) (15, 23). Because
intermedin is specifically expressed in the muscularis mucosae
layer of the stomach, it could have a role in gastrointestinal
functions. We therefore studied the ability of intermedin to
regulate gastric emptying activity in mice. As shown in Fig. 8,
intraperitoneal administration of intermedin suppressed gas-
tric emptying activity, similar to treatment with a known gas-
tric emptying suppression peptide, SRP/urocortin II (15, 23).
Likewise, treatment with ADM also suppressed gastric empty-
ing activity, but with a lower potency. Thus, intermedin could
mediate anorexic responses through the regulation of gastro-
intestinal motility (22, 24).
We have used a genomic approach to study novel polypeptide
ligands and receptors based on the evolutionary conservation of
polypeptides (14, 15, 25, 26). Based on the analysis of the
evolution of calcitonin/CGRP family ligands from diverse ver-
tebrates, we have identified a novel family peptide (intermedin)
that is expressed in the pituitary and digestive tract. Studies
using 293T cells expressing recombinant CRLR and RAMPs
demonstrated that intermedin is a bioactive peptide and acti-
vates Gs of the G protein family through CRLR/RAMP receptor
complexes. In contrast to CGRP and ADM, which exhibited a
preferential stimulation of CRLR when co-expressed with
RAMP1 and RAMP2 or RAMP3, respectively, intermedin rep-
resents a nonselective agonist for the three CRLR/RAMP re-
ceptor complexes.
Since the discovery of calcitonin in the 1960s, the calcitonin/
CGRP family peptides have been studied extensively. As a
result of gene duplication and functional divergence, this group
of peptide hormones acts on diverse systems. Coupled with two
closely related GPCRs and three unique RAMPs that transport
receptors to the cell surface, a complex receptor/ligand signal-
ing system operates in diverse vertebrates (5). Calcitonin,
CGRP, ADM, and amylin are expressed in a tissue-specific
manner, with the highest expression in thyroid C cells, the
central nervous system, adrenal glands, and islet B cells, re-
spectively. Although it has been recently established that the
signaling by CGRP, ADM, and amylin is unique among peptide
hormones and requires the formation of a receptor/RAMP com-
plex, the exact role of these peptides and their cognate recep-
tors in different physiologies remains to be investigated. The
present discovery of intermedin as a calcitonin/CGRP family
peptide highly expressed in the pituitary and digestive tract
provides a new ligand for peripheral regulation mediated by
the CRLR/RAMP system. As a first step in defining the role of
CRLR/RAMP receptor complexes in intermedin signaling, we
investigated the activation of CRLR/RAMP receptor complexes
in transfected 293T cells and demonstrated that RAMP is re-
quired for mediating intermedin action through CRLR. Of in-
7272 Intermedin Is a Novel Calcitonin/CGRP Family Peptide
FIG. 6. Decrease in systemic blood pressure and increase in
heart rate by intermedin and related peptides. A, dose-dependent
suppression of systolic blood pressure by IMDL, IMDS, and ADM in
male Sprague-Dawley rats. Blood pressure change was monitored for 40
min, and averages at 10, 20, and 30 min are presented. B, increase in
heart rate upon treatment with different doses of IMDL, IMDS, and
ADM in male Sprague-Dawley rats. C, blockage of the hypotensive
effect of intermedin by the CGRP receptor antagonist CGRP-(8 –37) and
the putative intermedin receptor-binding domain peptide IMD-(17– 47).
D, suppression of blood pressure in male SHRs by intermedin and
blockage of intermedin effects by receptor antagonists.
FIG. 7. Suppression of food intake by intermedin in fasted
mice. Shown is the cumulative food intake in mice treated with phos-
phate-buffered saline (n ⫽ 29), ADM (100 nmol/kg; n ⫽ 20), IMDS (100
nmol/kg; n ⫽ 16), IMDL (100 nmol/kg; n ⫽ 17), or the type II cortico-
tropin-releasing hormone receptor-selective agonist SRP/urocortin II
(100 nmol/kg; n ⫽ 10) (15, 23) at 1, 2, and 4 h after treatment.
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FIG. 8. Suppression of gastric emptying activity by interme-
din. Shown is the reduction of gastric emptying by IMDL (100 nmol/kg;
n ⫽ 24), IMDS (100 nmol/kg; n ⫽ 20), ADM (100 nmol/kg; n ⫽ 27), and
SRP/urocortin II (100 nmol/kg; n ⫽ 10) at 90 min after hormone treat-
ment compared with control animals receiving saline injections (n ⫽
31). Gastric emptying was calculated by comparing the stomach weight
of treated mice with the stomach weight of control mice receiving no
hormone treatment and killed at the time of hormone injection. Addi-
tional animals injected with saline and killed at the same time as
hormone-treated animals were used as experimental controls. *, signif-
icantly different from control animals injected with saline alone
(p ⬍ 0.05).
terest, intermedin exhibited a receptor activation profile dis-
tinct from that of CGRP or ADM, suggesting that intermedin
could be important for select CRLR/RAMP-mediated physiolog-
ical processes. It has been shown that the receptor activation
profiles of CGRP and ADM in native tissues are affected by
endogenous RAMPs present in different systems. Future stud-
ies on the interaction between intermedin and CRLR/RAMP
receptor complexes in different cell types and native tissues are
needed to clarify the importance of different RAMPs in inter-
medin physiology. Furthermore, it has been demonstrated that
even though CGRP and ADM overlap in receptor interaction,
each of these peptides apparently binds to unique binding
pockets (5); therefore, future studies on the structure-function
relationship of intermedin and related peptides are essential
for the characterization of the CRLR/RAMP-associated signal-
ing system.
Among calcitonin/CGRP family peptides, ADM is mainly
 Intermedin Is a Novel Calcitonin/CGRP Family Peptide
characterized as a hypotensive hormone (27–29), whereas
CGRP is important for sensory neurotransmission (3, 9 –11, 13,
30, 31). In addition, ADM inhibits bronchial constriction and
acts as a neurohormone to inhibit water drinking and salt
appetite (27, 32, 33). Studies using mutant mice suggest that
ADM is indispensable for vascular morphogenesis during em-
bryonic development (7, 8), whereas ␣-CGRP is important for
the modulation of sympathetic activity and inflammatory reac-
tions (9 –11). Therefore, CRLR in different tissues could medi-
ate the actions of multiple paralogous ligands, and the physi-
ological role of this receptor is partly dependent on activating
ligands derived from neighboring cells and/or the general cir-
culation. Because intermedin interacts with CRLR/RAMP re-
ceptor complexes, the known receptors for CGRP and ADM,
intermedin could regulate diverse physiological functions that
have been attributed to ADM or CGRP. As demonstrated in
this study, intermedin decreases blood pressure in both normal
rats and SHRs as effectively as the better characterized ADM
and CGRP, suggesting that intermedin could regulate vascu-
lature homeostasis (27). Immunohistochemistry studies have
shown that CRLR and RAMPs are found in the entire vascu-
lature and that CRLR is expressed mainly in the endothelial
layer (34); therefore, intermedin and related peptides decrease
blood pressure via the activation of CRLR/RAMP receptor com-
plexes in vascular endothelial cells. Concomitant with a hypo-
tensive effect, intermedin treatment also increases heart rate.
The increase in heart rate by intermedin and related peptides
could be a reflex response to the hypotensive effect; however,
the exact mechanisms remain to be investigated because the
CRLR gene has been shown to be expressed in cardiac myo-
cytes (35) in addition to the cardiac vasculature (34, 36). Fur-
thermore, intermedin could have cardioprotective and anti-
bronchial constriction activities that are important for the
regulation of cardiac and respiratory homeostasis (27, 37). Be-
cause intermedin is not detected in proximity to the vascula-
ture system using immunohistochemical analysis, further
studies are needed to reveal whether intermedin represents an
endocrine hormone involved in the regulation of cardiac and
vasculature systems.
Earlier studies on CGRP and ADM have shown that these
peptides and the CRLR signaling system play an important
role in gastrointestinal functions, including motility and secre-
tions from the stomach and colon (38, 39). Similar to earlier
studies on CGRP and ADM, exogenous intermedin administra-
tion was found to exhibit an anorexic effect and to suppress
stomach emptying responses in mice. These data suggest that
intermedin could have roles in the regulation of energy balance
via a paracrine mechanism; however, it is possible that the
observed effect on feeding behavior is secondary to alterations
in gastric motility. Because intermedin is expressed in multiple
gastrointestinal tissues, intermedin could have additional roles
in the gastrointestinal system that remain to be characterized.
In support of this view, it has been shown that CRLR is ex-
pressed in columnar cells lining the secretory ducts of the
parotid gland and in capillaries and venules of the esophagus
(34).
The observation that intermedin is expressed in the anterior
and intermediate lobes of the pituitary points to a potential role
for intermedin and the CRLR/RAMP signaling system in the
regulation of pituitary hormone secretion. Although the role of
intermedin in the regulation of pituitary hormone secretion
was not examined specifically in this study, earlier studies on
ADM have shown that administration of ADM increases circu-
lating prolactin levels in humans (40, 41). Therefore, it is
possible that intermedin may play a role in the regulation of
pituitary functions. Further studies on the exact expression
7273
pattern of intermedin in the pituitary and other tissues during
development are important for the understanding of interme-
din physiology.
Earlier studies on the evolution of peptide hormones have
shown that selection pressure has favored the conservation of
functionally important or mature regions of polypeptide hor-
mone precursors. The finding that only the C-terminal end of
the intermedin precursor was conserved during evolution sug-
gested that the C-terminal sequences of the intermedin precur-
sors represent the mature peptide and further strengthened
the theory that sequence conservation among species provides
important information on the functional characteristics of gene
sequences. Of interest, comparative sequence studies of inter-
medin precursors from different vertebrates showed that the
N-terminal cleavage sites of putative mature intermedins vary
in position, whereas a downstream arginine residue is com-
pletely conserved in all species studied. These data indicate
that mature intermedin from diverse species could be of vary-
ing lengths and that a shorter human intermedin (e.g. IMDS)
could be generated after post-translational processing at the
downstream basic residue. Future studies on human samples
are necessary to reveal the exact mature form(s) of human
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intermedin. Furthermore, the recent availability of multiple
sequenced vertebrate genomes will allow the identification and
characterization of additional peptide hormones on a global
scale based on the genomic profiling approach we used to iden-
tify intermedin and other novel peptide hormones (15).
In conclusion, we have identified and characterized a novel
calcitonin/CGRP family gene and demonstrated that encoded
intermedin peptides are biologically active in diverse in vitro and
in vivo CRLR/RAMP assays. Therefore, intermedin is a physio-
logical regulator of gastrointestinal, cardiovascular, and other
bioactivities mediated by the CRLR/RAMP receptor complexes.
Although the four mammalian CGRP-related peptide hormones
(␣-CGRP, ␤-CGRP, ADM, and intermedin) are capable of inter-
acting with CRLR, optimal regulation by this GPCR signaling
pathway likely depends on an integrated release of different
endocrine/paracrine ligands in a tissue-specific and time-coordi-
nated manner. Future studies on tissue distribution and endog-
enous fluctuation of intermedin under normal or pathological
conditions are important to formulate pharmacological therapies
for diverse pathological conditions in cardiovascular, pulmonary,
gastrointestinal, and neuroendocrine systems.
Acknowledgments—We thank Augustin Sanchez and Caren Spencer
for technical and editorial assistance. We gratefully acknowledge Drs.
Aaron J. W. Hsueh, Anita Payne, Linda Giudice, and Mary Lake Polan
(Department of Obstetrics and Gynecology, Stanford University School
of Medicine) for providing support and encouragement for these and
related efforts.
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 Intermedin Is a Calcitonin/Calcitonin Gene-related Peptide Family Peptide Acting
through the Calcitonin Receptor-like Receptor/Receptor Activity-modifying Protein
Receptor Complexes
Jaesook Roh, Chia Lin Chang, Alka Bhalla, Cynthia Klein and Sheau Yu Teddy Hsu
J. Biol. Chem. 2004, 279:7264-7274.
doi: 10.1074/jbc.M305332200 originally published online November 13, 2003

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